Chemokines are a superfamily of small, cytokine-like proteins that induce cytoskeletal rearrangement, firm adhesion to endothelial cells, and directional migration, and may also effect cell activation and proliferation. Chemokines act in a coordinated fashion with cell surface proteins to direct the specific homing of various subsets of cells to specific anatomical sites. Chemokines are thought to mediate their effect by binding to seven-transmembrane G protein-coupled receptors to attract leukocyte subsets to sites of inflammation, and are generally thought to play an important role in the initiation and maintenance of inflammation (Baglionini et al. (1998) Nature 392: 565-568); Luster, A. D., New Eng. J. Med., (1998) 338(7), 436).
Stromal cell derived factor one (SDF-1) is a member of the CXC family of chemokines. SDF-1 has two isoforms (SDF-1α and SDF-1β); Genbank accession numbers L36033 and L36034, respectively), which are closely related, and is referred to herein collectively as SDF-1. SDF-1 is the natural ligand for the chemokine receptor CXCR4. CXCR4 receptor was originally cloned by Loetscher et al. (see, Loetscher, M., et al. J. Biol. Chem. (1994) 269, 232) and is found to be expressed on neutrophils, monocytes, myeloid cells and T lymphocytes (see, Murdoch, C., et al. Blood, (2000) 95(10), 3032).
CXCR4 has been found to be the major co-receptor for T-tropic HIV-1 entry, i.e., it interacts with HIV and with the cellular CD4 receptor to facilitate viral entry into cells (See, Feng, Y., et al. Science (1996) 272, 872). In addition to functioning as a HIV entry co-receptor, the CXCR4 receptor plays a role in many cell signaling processes. The importance of the signaling mechanism between the CXCR4 natural ligand, pre-B-cell growth-stimulating factor/stromal cell derived factor (PBSF/SDF-1) to the CXCR4 chemokine receptor has been described by researchers. CXCR4 receptor has been found to be essential for the vascularization of the gastrointestinal tract (Tachibana, et al., Nature (1998) 393:591-594) as well as haematopoiesis and cerebellar development (Zou, et al., Nature (1998) 393:591-594). Interference with any of these important functions served by the binding of pre-B-cell growth-stimulating factor/stromal derived factor (PBSF/SDF-1) to the CXCR4 chemokine receptor results in lethal deficiencies in vascular development, haematopoiesis and cardiogenesis.
SDF-1 is functionally distinct from other chemokines in that it is reported to have a fundamental role in the trafficking, export and homing of bone marrow progenitor cells (See, Hattori, K., et al. Blood (2000) 97, 3354-3360; WO 2005/000333). It has been reported that CXCR4-deficient mice display hematopoietic defects (Nagasawa et al. Nature (1996) 382, 635-638). The migration of CXCR4 expressing leukocytes and hematopoietic progenitors to SDF-1 appears to be important for maintaining B-cell lineage and localization of CD34+ progenitor cells in bone marrow (see, Bleul et al. J. Exp. Med. (1998) 187, 753-762; Viardot et al. Ann. Hematol. (1998) 77, 195-197; Auiti et al. J. Exp. Med. (1997) 185, 111-120; Peled et al. Science (1999) 283, 845-848; Qing et al. Immunity (1999) 10, 463-471; Lataillade et al. Blood (1999) 95, 756-768; Ishii et al. J. Immunol. (1999) 163, 3612-3620; Maekawa et al. Internal Medicine (2000) 39, 90-100; Fedyk et al. J. Leukocyte Biol. (1999) 66, 667-673; Peled et al. Blood (2000) 95, 3289-3296). Particularly high levels of SDF-1 are found in bone-marrow stromal cells (Shirozu, M. et al. (1995) Genomics, 28, 495-500; Bleul, C. C. et al., (1996) J. Exp. Med. 184, 1101-1109).
The cell signaling initiated upon binding of SDF-1 to CXCR4 may also play an important role in tumor cell proliferation and regulation of angiogenesis associated with tumor growth (See, “Chemokines and Cancer” published by Humana Press (1999), Edited by B. J. Rollins; Arenburg et al. J Leukocyte Biol. (1997) 62, 554-562; Moore et al. J. Invest. Med. (1998) 46, 113-120; Moore et al. Trends Cardiovasc. Med. (1998) 8, 51-58; Seghal et al. J. Surg. Oncol. (1998) 69, 99-104). The expression of CXCR4 has been associated with osteosarcoma, pancreatic cancer, brain, breast, and colon cancer. (See, Paoletti et al. Int J. Oncol; (2001) 18, 11-6); Koshiba et al. Clin Cancer Res. (2000) 6, 3530-5); Muller, et al., Nature (2001) 410:50-56; and Murphy et al., WO 99/50461). In addition, some chemokines have been linked to metastasis of cancer from specific organs, including lymph node, bone marrow, and skin; or from carcinomas of breast, head and neck, melanoma or prostate origin. (See, Mueller et al., WO 01/38352).
The SDF-1/CXCR4 cell signaling pathway have been found to be involved in modulating non-tumor associated angiogenesis. Mice deficient for either CXCR4 or SDF-1 have defects in the formation of the large blood vessels that supply the organs of the GI tract and the brain, see Yong-Rui Zou, et. al., Nature 393, 591-594 (1998); Kazunobu Tachibana et. al., Nature 393, 595-599 (1998) and Takashi Nagasawa et. al., Nature 382, 635-638 (1996). In addition, subcutaneous injection of SDF-1 causes localized neovascularization (Rosalba Salcedo et al., American Journal of Pathology 154: 1125-1135 (1999)).
A role for CXCR4 in ocular neovascular disease is suggested by its expression pattern in the eye. mRNA for CXCR4 has been shown to “be expressed in vascular endothelial cells that are a component of blood vessels and capillaries (Orribretta Salvucci et al., Blood 99: 2703-2711 (2002). In addition, CXCR4 is expressed in the retinal pigmented epithelium (RPE) that lies between the choroidal vasculature and the retinal neurons (Isabel Crane et al., Journal of Immunology 165: 4372-4378 (2000). Thus, CXCR4 is in the right location to influence the process of CNV and diabetic retinopathy. It is also possible that CXCR4 may play a role in the non-neovascular form of AMD, also called dry or atrophic AMD. There is evidence to suggest that inflammation may contribute to the pathogenesis of dry AMD (Philip Penfold et al., Progress in Retinal and Eye Research 20: 385-414 (2001), and CXCR4 has been implicated in the inflammatory process (Nicholas Lukacs et al., American Journal of Pathology 160: 1353-1360 (2002); Patrick Matthys et al., Journal of Immunology 167: 4686-4692 (2001) and Jose-Angel Gonzalo et al., Journal of Immunology 165: 499-508 (2000).
The interaction of SDF-1 to CXCR4 has also been implicated in the pathogenesis of atherosclerosis (Abi-Younes et al. Circ. Res. 86, 131-138 (2000)), renal allograft rejection (Eitner et al. Transplantation 66, 1551-1557 (1998)), asthma and allergic airway inflammation (Yssel et al. Clinical and Experimental Allergy 28, 104-109 (1998); J. Immunol. 164, 5935-5943 (2000); Gonzalo et al. J. Immunol. 165, 499-508 (2000)), Alzheimer's disease (Xia et al. J. Neurovirology 5, 32-41 (1999)), rheumatoid arthritis (US 2005/0202005) and Arthritis (Nanki et al. J. Immunol. 164, 5010-5014 (2000)).
As described above, early research efforts by a number of groups have indicated a role for the chemokine receptor CXCR4 in metastasis and tumor growth. Muller, et al., “Involvement of Chemokine Receptors in Breast Cancer Metastasis,” Nature, 410:50-56 (2001) demonstrated that breast tumor cells use chemokine-mediated mechanisms, such as those regulating leukocyte trafficking, during the process of metastasis. Tumor cells express a distinct, non-random pattern of functionally active chemokine receptors. Signaling through CXCR4 mediates actin polymerization and pseudopodia formation in breast cancer cells, and induces chemotactic and invasive responses. Additionally, the organs representing the main sites of breast cancer metastasis (such as lymph nodes, bone marrow, and lungs) are the most abundant sources of ligand for the CXCR4 receptor.
Using immunodeficient mice, Muller and colleagues succeeded in reducing the metastasis of injected human breast cancer cells by treating mice with an antibody known to bind CXCR4. Their finding suggests that breast cancer metastasis could be reduced by treating a patient with a CXCR4 antagonist.
Bertolini, et al., “CXCR4 Neutralization, a Novel Therapeutic Approach for Non-Hodgkin's Lymphoma,” Cancer Research, 62:3106-3112 (2002) demonstrated a reduction of tumor volume as well as prolonged survival of immunodeficient mice injected with human lymphoma cells treated with anti-CXCR4 antibodies. They interpreted their finding to mean that tumor volume could be reduced by treating a patient with a CXCR4 antagonist.
More recent studies suggest that another chemokine receptor, CCXCKR2, may also be a potential candidate in the treatment of cancer. CCXCKR2 is preferentially expressed in transformed cells over normal cells, with detectable expression in a number of human cancers. In vitro studies indicate that proliferation of CCXCKR2 expressing cells can be inhibited by an antagonist of CCXCKR2. In vivo studies in mice indicate that CCXCKR2 antagonists can inhibit tumor formation and tumor growth.
The potential importance of CCXCKR2 is illustrated by an alternative interpretation of the reduction in tumor volume seen by Bertolini and colleagues. This reduction could clearly be the result of an antibody-mediated clearance, and not the result of the anti-CXCR4 antibody as originally believed. In an antibody-mediated clearance, any antibody that recognized a protein on the cell surface of the lymphoma cells would have had the same effect as that attributed to the anti-CXCR4 antibody. Unfortunately, Bertolini and colleagues studies are inconclusive as to whether the observed tumor response was due to antibody-mediated clearance or interaction with CXCR4.
However it is now known that the lymphoma cells used by Bertolini and colleagues express both CXCR4 and CCXCKR2. SDF-1 is the only ligand for CXCR4. SDF-1 and I-TAC both bind CCXCKR2. Using anti-SDF-1 antibody, it has now been shown that antagonists of CCXCKR2 are responsible for the reduction in tumor load and increased survival rate. Because SDF-1 is the only ligand for CXCR4, one would expect neutralization of SDF-1 with anti-SDF-1 antibody would be equivalent to the neutralization of CXCR4 with anti-CXCR4 antibody. However, experiments using an anti-SDF-1 antibody demonstrated only a partial reduction in tumor load and an increased survival rate. As a result, CCXCKR2 is the likely target, as the continued activity appears due to the interactions of the second ligand, I-TAC, with CCXCKR2.
Until recently, the possible importance of CCXCKR2 in tumor cell proliferation, tumor growth, and metastasis was unknown. Now, with recent evidence pointing to the ability of certain CCXCKR2 antagonists to prevent the growth and spread of cancer, and expression patterns indicating a limited tissue distribution for the CCXCKR2 receptor.
Moreover, recently it has been discovered that CCXCKR2 can serve as a co-receptor for certain genetically divergent human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), in particular for the HIV-2-ROD, an X4-tropic isolate (Shimizu, N. et al., J. Virol., (2000) 74: 619-626; Balabanian, K., et al., J. Biol. Chem., in press; published on Aug. 17, 2005 as Manuscript M508234200).
Still further, SDF-1, has been described to have a role in the mobilization of hematopoietic progenitor cells and stem cells, and in particular of those cells bearing the CXCR4 receptor, to specific hematopoietic tissues including bone marrow has been described (Hattori, K., et al., Blood, (2000) 97:3354-3360; WO 2005/000333, the disclosure of which are incorporated herein by reference). For example, it is known that CD34+ progenitor cells express CXCR4 and require SDF-1 produced by bone marrow stromal cells for chemoattraction and engraftment, and that in vitro, SDF-1 is chemotactic for both CD34+ cells and for progenitor/stem cells. SDF-1 is also an important chemoattractant, signaling via the CXCR4 receptor, for several other more committed progenitors and mature blood cells including T-lymphocytes and monocytes, pro- and pre-B lymphocytes, and megakaryocytes. As mentioned above, SDF-1 is the only ligand for the CXCR4 receptor. SDF-1 and I-TAC are both ligands for CCXCKR2 receptor. More recent studies suggest that the CCXCKR2 receptor may also play a part in stem cell mobilization processes.
It is apparent that the CXCR4 chemokine receptor and associated cell signaling processes plays a role in the pathology diseases, such as cancer, inflammation, HIV, stem cell related disorders, ocular disorders, etc. As such, it would be beneficial to have small molecule inhibitors of the CXCR4 receptor, that could serve to modulate (e.g., antagonize, agonize) the binding, signaling and chemotactic effects of the SDF-1 for the receptor. CXCR4. In addition, in view of the above, it is also apparent that compounds that are able to bind specifically to CCXCKR2 receptors may be useful to treating diseases and other biological conditions that may benefit from such interactions. The present invention fulfills this and other needs.